Hydrodynamic focusing apparatus and methods

ABSTRACT

A microfluidic chip having a micro channel for processing a sample is provided. The micro channel may focus the sample by using focusing fluid and a core stream forming geometry. The core stream forming geometry may include a lateral fluid focusing component and one or more vertical fluid focusing components. A microfluidic chip may include a plurality micro channels operating in parallel on a microfluidic chip.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/213,800, filed Mar. 14, 2014, which claims the benefit of priority toU.S. Provisional Application Ser. No. 61/785,734, titled “HydrodynamicFocusing Apparatus and Methods,” and filed Mar. 14, 2013, the contentsof each of the above applications is hereby incorporated by reference inits entirety.

TECHNICAL FIELD

Generally, this disclosure relates to hydrodynamic focusing, inparticular, in a microfluidic device. More specifically, the presentdiscloser relates to systems and methods for producing a sheath flow ina flow channel and, in particular, in a micro channel in a microfluidicdevice.

BACKGROUND

Sheath flow is a particular type of laminar flow in which one layer ofsample fluid, or a particle, is surrounded by another layer of focusingfluid on more than one side. The process of confining a particle streamin a fluid is referred to as a ‘sheath flow’ configuration. For example,in a sheath flow configuration, a sheath fluid may envelop and pinch asample fluid containing a number of particles. The flow of the samplefluid containing particles suspended therein may be narrowed almost tothe outer diameter of particles in the center of the sheath fluid. Theresulting sheath flow flows in a laminar state within an orifice orchannel so that the particles are aligned and accurately pass through anorifice or channel in a single file row.

Sheath flow is used in many applications where it is preferable toprotect particles or fluids by a layer of sheath fluid, for example inapplications wherein it is necessary to protect particles from air. Forexample, in particle sorting systems, flow cytometers and other systemsfor analyzing a sample, particles to be sorted or analyzed are usuallysupplied to a measurement position in a central fluid current, which issurrounded by a particle free liquid sheath.

Sheath flow is useful because it can position particles with respect tosensors or other components and prevent particles in the center fluid,which is surrounded by the sheath fluid, from touching the sides of theflow channel and thereby prevents clogging of the channel. Sheath flowallows for faster flow velocities and higher throughput of samplematerial. Faster flow velocity is possible without shredding cells inthe center fluid because the sheath fluid protects the cells frompotentially high shear forces at the walls of the flow channel.

Conventional devices that have been employed to implement sheath flowhave relatively complex designs and are relatively difficult tofabricate.

SUMMARY

According to aspects of the disclosure, a microfluidic particleprocessing assembly including a substrate and a flow channel formed inthe substrate may be provided. The flow channel may include an inlet, afluid focusing region having an associated fluid focusing feature forfocusing a particle within the flow channel, and an inspection region atleast partially downstream of the fluid focusing region. Further, theflow channel may have first and second outlets.

According to other aspects, the fluid focusing features of the flowchannel focusing region may include a core stream forming geometry. Thecore stream forming geometry may further include a lateral fluidfocusing region, a first vertical fluid focusing component, and a secondvertical fluid focusing component.

According to some aspects, the first vertical fluid focusing componentmay include a vertical fluid focusing channel and the second verticalfluid focusing component may include a second vertical fluid focusingchannel. The first vertical fluid focusing component and the secondvertical fluid focusing component may be in communication with the fluidfocusing region in opposite vertical directions. The first verticalfluid focusing component may provide a first vertical influence and thesecond vertical fluid focusing component may provide a second verticalinfluence in the opposite directions as the first vertical influence.

According to other aspects, the flow channel may further include asheath inlet in fluid communication with the sheath source. A sampleinlet may be positioned within a sheath flow created by the sheath inletto facilitate a co-axial flow of sheath and sample. The sample inlet mayinclude a tapered or beveled inlet.

According to yet other aspects, the flow channel may have a first widthand a first height at the sample inlet. The flow channel may a secondwidth and a second height at a first transition point. The height of theflow channel may be reduced between the sample inlet and the firsttransition point. The flow channel may have a third width and a thirdheight at a second transition point. The height of the flow channel mayremain constant between the first transition point and the secondtransition point and the width of the flow channel may be reducedbetween the first transition point and the second transition point. Thethird height and the third width of the flow channel may be maintainedthrough the inspection region. The flow channel may transition from asquare cross section to a rectangular cross section. The flow channelmay transition from a circular cross section to an elliptical crosssection.

The microfluidic assembly may further include a plurality of flowchannels as presented herein.

According to other aspects, the fluid focusing feature of the fluidfocusing region may further include ultrasonic transducers for producingpressure waves in the focusing region of each flow channel. Theultrasonic transducers may be an array of ultrasonic transducers forproducing a standing pressure wave along the flow channel.

According to even other aspects, a diverting mechanism in communicationwith the flow channel may be provided. The diverting mechanism mayinclude a bubble valve. Alternatively, the diverting mechanism mayinclude an array of ultrasonic and/or standing acoustic wavetransducers. Optionally, the diverting mechanism may includeinterdigitated transducers (IDT).

According to certain aspects, a microfluidic chip may include asubstantially planar chip substrate having an upper surface and a lowersurface. A microfluidic flow channel may be provided within the chipsubstrate. A first inlet port may be formed on the upper surface of thechip substrate for receiving a focusing fluid. The first inlet port maybe in fluid communication with the microfluidic flow channel. Themicrofluidic flow channel may include a first focusing fluid inletconfigured to introduce focusing fluid from the first inlet port intothe microfluidic channel in a first direction, a second focusing fluidinlet configured to introduce focusing fluid from the first inlet portinto the microfluidic channel in a second direction, and a thirdfocusing fluid inlet configured to introduce focusing fluid from thefirst inlet port into the microfluidic channel in a third direction.

According to certain other aspects, the microfluidic chip may alsoinclude a second inlet port formed on the upper surface of the chipsubstrate for receiving a focusing fluid. The second inlet port may bein fluid communication with the microfluidic flow channel. Themicrofluidic flow channel may include a fourth focusing fluid inletconfigured to introduce focusing fluid from the second inlet port intothe microfluidic channel in a fourth direction. The second focusingfluid inlet may be configured to introduce focusing fluid from thesecond inlet port into the microfluidic channel in the second direction,and the third focusing fluid inlet may be configured to introducefocusing fluid from the second inlet port into the microfluidic channelin the third direction.

The microfluidic flow channel may include a fluid flow focusing regionhaving an upstream end region and a downstream end region. The firstfocusing fluid inlet may be configured to introduce focusing fluid intothe fluid flow focusing region in the upstream end region. The secondand third focusing fluid inlets may be configured to introduce focusingfluid into the fluid flow focusing region in the downstream end region.

According to other aspects, a microfluidic chip may include asubstantially planar substrate having an upper surface and a lowersurface. A microfluidic channel may be formed in the substantiallyplanar substrate and may have an upper surface and a lower surface. Aninlet port may be formed on the upper surface of the substantiallyplanar substrate and may be configured to receive a focusing fluid. Afirst focusing fluid channel in fluid communication with the inlet portmay be provided. The first focusing fluid channel may be configured tointroduce focusing fluid into the microfluidic channel via a firstaperture in the upper surface of the microfluidic channel. A secondfocusing fluid channel in fluid communication with the inlet port may beprovided. The second focusing fluid channel may be configured tointroduce focusing fluid into the microfluidic channel via a secondaperture in the lower surface of the microfluidic channel.

The microfluidic channel and the first and second focusing fluidchannels may be formed when a lower surface of an upper substrate layerand an upper surface of a lower substrate layer are joined together.

The microfluidic channel may lie in a first plane upstream of the firstaperture and in a second plane downstream of the second aperture.

At least one outlet port may be formed on the upper surface of thesubstantially planar substrate and in fluid communication with the fluidflow focusing region.

Certain embodiments of the disclosed apparatus and methods aresummarized below. These embodiments are not intended to limit the scopeof the disclosure, but rather serve as brief descriptions of exemplaryembodiments. Both the disclosure and claimed invention may encompass avariety of forms which differ from these summaries.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the present disclosure are further describedwith reference to the appended figures. It is to be noted that thevarious features and combinations of features described below andillustrated in the figures can be arranged and/organized differently toresult in embodiments which are still within the spirit and scope of thepresent disclosure. To assist those of ordinary skill in the art inmaking and using the disclosed systems, assemblies and methods,reference is made to the appended figures.

FIG. 1 schematically illustrates an exemplary particle processing systemaccording to the present disclosure.

FIG. 2 illustrates an exemplary microfluidic chip according to thepresent disclosure;

FIG. 3A is a top perspective view of a portion of a flow channelgeometry with arrows schematically depicting flow of sample fluid andfocusing fluid in accordance with certain embodiments described herein.

FIG. 3B is a bottom perspective view of a portion of a flow channelgeometry in accordance with the embodiment of FIG. 3A, with arrowsschematically depicting flow of sample fluid and focusing fluid inaccordance with certain embodiments described herein.

FIG. 3C is top view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 3A.

FIG. 3D is a cross-section through line 3D-3D of FIG. 3C of a portion ofa flow channel geometry in accordance with the embodiment of FIG. 3A.

FIG. 3E is bottom view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 3A.

FIG. 4A is a top perspective view of a portion of a flow channelgeometry with arrows schematically depicting flow of sample fluid andfocusing fluid in accordance with certain embodiments described herein.

FIG. 4B is top view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 4A.

FIG. 4C is a cross-section through line 4C-4C of FIG. 4B of a portion ofa flow channel geometry in accordance with the embodiment of FIG. 4A.

FIG. 4D is bottom view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 4A.

FIG. 5A is a top perspective view of a portion of a flow channelgeometry with arrows schematically depicting flow of sample fluid andfocusing fluid in accordance with certain embodiments described herein.

FIG. 5B is top view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 5A.

FIG. 5C is a cross-section through line 5C-5C of FIG. 5B of a portion ofa flow channel geometry in accordance with the embodiment of FIG. 5A.

FIG. 5D is bottom view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 5A.

FIG. 6A is a top perspective view of a portion of a flow channelgeometry with arrows schematically depicting flow of sample fluid andfocusing fluid in accordance with certain embodiments described herein.

FIG. 6B is top view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 6A.

FIG. 6C is a cross-section through line 6C-6C of FIG. 6B of a portion ofa flow channel geometry in accordance with the embodiment of FIG. 6A.

FIG. 6D is bottom view of a portion of a flow channel geometry inaccordance with the embodiment of FIG. 6A.

FIG. 7A is top view of a portion of a substrate of the microfluidicchip, schematically illustrating micro channel geometry, in accordancewith the embodiment of FIG. 5A, formed between an upper substrate layerand a lower substrate layer.

FIG. 7B is bottom view of a portion of an upper substrate layer inaccordance with the embodiment of FIGS. 5A and 7A, schematicallyillustrating the micro channel geometry formed in the lower surface ofthe upper substrate layer.

FIG. 7C is top view of a portion of a lower substrate layer inaccordance with the embodiment of FIGS. 5A and 7A, schematicallyillustrating the micro channel geometry formed in the upper surface ofthe lower substrate layer.

While the present disclosure may be embodied with various modificationsand alternative forms, specific embodiments are illustrated in thefigures and described herein by way of illustrative examples. It shouldbe understood the figures and detailed descriptions are not intended tolimit the scope of the claims to the particular form disclosed, but thatall modifications, alternatives, and equivalents falling within thespirit and scope of the claims are intended to be covered.

DETAILED DESCRIPTION

A microfluidic particle (e.g., cell) analysis and/or sorting system fora microfluidic chip, in accordance some embodiments, may have a widevariety of applications as a therapeutic medical device enablingcell-based therapies, such as blood transfusion, bone marrowtransplants, and/or mobilized peripheral blood implants. Embodiments ofmicrofluidic sorting systems may be capable of selecting cells based onintrinsic characteristics as determined by interaction of light with thecells (e.g., scatter, reflection, and/or auto fluorescence) independentof protocols and necessary reagents. A microfluidic system may employ aclosed, sterile, disposable cartridge including a microfluidic chip. Themicrofluidic system may process particles (e.g., cells) at high speeds,and deliver particles (e.g., cells) with high yield and high purity.

Certain embodiments described herein relate systems and methods forproducing a sheath flow in a flow channel and, in particular, in a microchannel in microfluidic devices.

As used herein, the term “particles” includes, but is not limited to,cells (e.g., blood platelets, white blood cells, tumorous cells,embryonic cells, spermatozoa, etc.), synthetic beads (e.g.,polystyrene), organelles, and multi-cellular organisms. Particles mayinclude liposomes, proteoliposomes, yeast, bacteria, viruses, pollens,algae, or the like. Particles may also refer to non-biologicalparticles. For example, particles may include metals, minerals,polymeric substances, glasses, ceramics, composites, or the like.Additionally, particles may include cells, genetic material, RNA, DNA,fragments, proteins, etc. or bead, for example, with fluorochromeconjugated antibodies.

As used herein, the term “microfluidic system” refers to a system ordevice including at least one fluidic channel having microscaledimensions. The microfluidic system may be configured to handle,process, detect, analyze, eject, and/or sort a fluid sample and/orparticles within a fluid sample. The term “channel” as used hereinrefers to a pathway formed in or through a medium that allows formovement of fluids, such as liquids and gases. The term “micro channel”refers to a channel, preferably formed in a microfluidic system ordevice, having cross-sectional dimensions in the range between about 1.0μm and about 2000 preferably between about 25 μm and about 500 and mostpreferably between about 50 μm and about 300 μm. One of ordinary skillin the art will be able to determine an appropriate volume and length ofthe micro channel for the desired application. The ranges above areintended to include the above-recited values as upper or lower limits.The micro channel may have any selected cross-sectional shape orarrangement, non-limiting examples of which include a linear ornon-linear configuration, a U-shaped or D-shaped configuration, and/or arectangular, triangular, elliptical/oval, circular, square, ortrapezoidal geometry. A microfluidic device or microfluidic chip mayinclude any suitable number of micro channels for transporting fluids. Amicrofluidic chip may be provided as a disposable cartridge with aclosed channel system.

As used herein the terms “vertical,” “lateral,” “top,” “bottom,”“above”, “below,” “up,” “down,” and other similar phrases should beunderstood as descriptive terms providing general relationship betweendepicted features in the figures and not limiting on the claims,especially relating to flow channels and microfluidic chips describedherein, which may be operated in any orientation.

The present disclosure bears relations to U.S. Pat. No. 7,311,476 whichis hereby incorporated by reference.

Referring now to FIG. 1, a particle processing system 200 may beconfigured, dimensioned and adapted for analyzing, sorting, and/orprocessing (e.g., purifying, measuring, isolating, detecting, monitoringand/or enriching) particles (e.g., cells, microscopic particles, etc.)or the like. For example, system 200 may be a cytometer and/or a cellpurification system or the like, although the present disclosure is notlimited thereto. Rather, system 200 may take a variety of forms, and itis noted that the systems and methods described may be applied to otherparticle processing systems.

In exemplary embodiments, system 200 is a microfluidic flow sorterparticle processing system (e.g., microfluidic chip based system) or thelike. Exemplary microfluidic flow sorter particle processing systems andcomponents or the like are disclosed, for example, in U.S. Pat. No.8,529,161 (Ser. No. 13/179,084); U.S. Pat. No. 8,277,764 (Ser. No.11/295,183); U.S. Pat. No. 8,123,044 (Ser. No. 11/800,469); U.S. Pat.No. 7,569,788 (Ser. No. 11/101,038); U.S. Pat. No. 7,492,522 (Ser. No.11/906,621) and U.S. Pat. No. 6,808,075 (Ser. No. 10/179,488); and USPatent Publication Nos. 2012/0277902 (Ser. No. 13/342,756); 2011/0196637(Ser. No. 13/022,525) and 2009/0116005 (Ser. No. 12/259,235); and U.S.Patent Application Ser. No. 61/647,821 (Ser. No. 13/896,213) and61/702,114 (Ser. No. 14/029,485), 61/784,323, the foregoing beingincorporated herein by reference in their entireties.

In further exemplary embodiments, system 200 may be a multi-channel ormulti-jet flow sorter particle processing system (e.g., multiplecapillaries or multiple fluid jet-based systems) or the like. Exemplarymulti-channel or multi-jet flow sorter particle processing systems andcomponents or the like are disclosed, for example, in US PatentPublication No. 2005/0112541 (Ser. No. 10/812,351), the entire contentsof which is hereby incorporated by reference in its entirety.

FIG. 1 illustrates a system 200 suitable for implementing anillustrative embodiment of the present disclosure. System 200 includes amicrofluidic assembly 220. Microfluidic assembly 220 includes and/or isin communication with a particle inspection region and a sample fluidinput region. Microfluidic assembly 220 may include a plurality of microchannels for conveying a substance, such as particles or cells,therethrough. In certain embodiments and as can be understood by thosefamiliar in the art, microfluidic assembly 220 may be a combination ofmicrofluidic chips, micro channels, cuvettes, capillaries, nozzles, orjets which may combine to produce a multichannel particle processingsystem. The micro channels transport fluid and/or particles through theassembly 220 for processing, handling, and/or performing any suitableoperation (e.g., on a liquid sample). Assembly 220 may include anysuitable number of micro channels for transporting fluids throughassembly 220.

In exemplary embodiments, an optical detector system 226 for use withmicrofluidic assembly 220 may be provided. Optical detector system 226may be configured for the interrogation of the particles flowing throughor located within an interrogation region. Further, optical detectorsystem 226 may monitor flow through a plurality of channelssimultaneously. In exemplary embodiments, system 226 can inspectindividual particles for one or more particular characteristics, such assize, form, fluorescence, optical scattering, as well as othercharacteristics.

System 200 also includes at least one electromagnetic radiation or lightsource 221 (e.g., a laser source or the like) for simultaneously orsequentially illuminating at least a portion of each of an interrogationregion. The electromagnetic radiation source 221 may be coupled toand/or in communication with beam shaping optics 225 (e.g., segmentedmirror/mirrors or the like) for producing and forming a beam ofelectromagnetic radiation (e.g., light) 227. The light source 221 may beprovide as one or more monochromatic light sources, polychromatic lightsources, or any combination of the aforementioned. In general, theelectromagnetic radiation source(s) 221 may have any suitable wavelengthand one skilled in the art will recognize that any suitable lightsource(s) may be used.

In some embodiments, the one or more radiation beams 227 may passthrough an optical mask aligned with a plurality of particle-conveyingmicro channels in the microfluidic assembly 220. The optical mask maytake the form of an array of pinholes (e.g., provided in an opticallyopaque layer) associated with the interrogation regions of the pluralityof micro channels. Other spatial and/or spectral filter arrays may beprovided in the illumination and/or detection path of the particleprocessing system 200.

Examples of optical signals that may be produced in optical particleanalysis, cytometry and/or sorting when a beam 227 intersects a particleinclude, without limitation, optical extinction, angle dependent opticalscatter (forward and/or side scatter) and fluorescence. Opticalextinction refers to the amount of electromagnetic radiation or lightthat a particle extinguishes, absorbs, or blocks. Angle dependentoptical scatter refers to the fraction of electromagnetic radiation thatis scattered or bent at each angle away from or toward the incidentelectromagnetic radiation beam. Fluorescent electromagnetic radiationmay be electromagnetic radiation that is absorbed and/or scattered bymolecules associated with a particle or cell and re-emitted at adifferent wavelength. In some instances, fluorescent detection may beperformed using intrinsically fluorescent molecules.

In exemplary embodiments, optical detector system 226 may include one ormore detector subsystems 230 to capture and observe the optical signalsgenerated by the intersection of electromagnetic radiation beam 227 witha particle in a channel. Detector subsystems 230 may include one or moreextinction detector assemblies 231 for capturing extinction signals, oneor more scatter detector assemblies 233 for capturing scatter signals,and one or more fluorescence detector assemblies 235 for capturingfluorescence signals. In a preferred embodiment, detector system 226 mayinclude at least one extinction detector assembly 231, at least onescatter detector assembly 233, and at least one fluorescence detectorassembly 235. Detector assemblies 231, 233, 235 may includephotomultipliers, photodiodes, cameras, or other suitable device(s).

According to certain aspects, a detector subsystem 230 may include oneor more micro-lens systems 250. A plurality of micro-lens systems 250may be provided as a micro-lens array 260. Further, detector subsystems230 may include fiber optics or other waveguide-type opticaltransmission elements to direct the signals to the sensor elements, oneor more lenses, filters, mirrors, and/or other optical elements tocollect, shape, transmit, etc. the signal exiting the interrogationregion 222 and being received by the detector subsystems 230.

According to certain embodiments, a single detector subsystem 230 may beassociated with a plurality of interrogation sites (e.g., microfluidicchannels) and thus, may receive signals (simultaneously, sequentially,overlapping, non-overlapping, etc.) from each of the plurality ofinterrogation sites. The detector subsystems 230 may be connected toelectronics (not shown) to analyze the signals received from thedetector assemblies and/or control one or more aspects of the particlesorting system 200.

According to certain embodiments and referring to FIG. 2, microfluidicassembly 220 may be configured as a microfluidic chip 20 and may includea substrate 21 having a plurality of channels 30 (e.g., micro channels)disposed or formed therein. The micro channels 30 may be configured totransport fluid and/or particles through the microfluidic chip 20 forprocessing, handling, and/or performing any suitable operation on aliquid sample (e.g., a particle sorting system). For example, each microchannel 30 may be a flow cytometer. According to certain aspects, themicro channels 30 may be arranged parallel to each other.

As best shown in FIG. 2, the microfluidic chip 20 may include an inputregion 24 in which a sample containing particles (e.g., cells, etc.) isinput into the microfluidic chip 20 for processing and an output region26 for removing the processed sample from the microfluidic chip 20. Thesubstrate 21 may be provided as a substantially planar substrate, i.e.,having a first dimension (e.g., thickness t) much less than its othertwo dimensions (e.g., length L and width W). Further, the substrate 21of the microfluidic chip 20 may include first and second major planesurfaces: an upper surface 21 a; and a lower surface 21 b.

The sample fluid may be input via a sample inlet port 410 through theupper surface 21 a of the microfluidic chip 20. Each micro channel 30may have an interrogation region 222 associated therewith. Particles inchannels 30 may be detected while flowing through the interrogationregions 222. At the interrogation region 222, individual particles maybe inspected or measured for a particular characteristic, such as size,form, orientation, fluorescence intensity, etc. Interrogation regions222 may be illuminated through the upper surface 21 a and/or the lowersurface 21 b of the microfluidic chip 20.

The plurality of channels 30 may be evenly distributed (i.e., evenlyspaced) across the width W of the microfluidic chip 20. According tocertain embodiments, a centerline-to-centerline spacing between thechannels 30 may range from 0.2 mm to 5.0 mm. Thecenterline-to-centerline spacing between the micro channels 30 may beless than 4.0 mm, less than 3.0 mm, or even less than 1.0 mm. Accordingto certain embodiments, the centerline-to-centerline spacing between themicro channels 30 may range from 2.5 mm to 3.0 mm. Advantageously, tominimize the footprint of the microfluidic chip 20, thecenterline-to-centerline spacing between the micro channels 30 may beless than 2.0 mm, less than 1.5 mm, or even less than 1.0 mm. Accordingto certain embodiments, the centerline-to-centerline spacing between themicro channels 30 may range from 0.7 mm to 1.2 mm.

The substrate 21 of the microfluidic chip 20 may be formed with one ormore substrate layers 60. As shown in FIG. 2, the substrate 21 may beformed by bonding or otherwise attaching an upper substrate layer 62 toa lower substrate layer 64. In general, any number of layers may be usedto form microfluidic chip 20.

The substrate layers 60 of the microfluidic chip 20 may be glass (e.g.,UV fused-silica, quartz, borofloat, etc.), PDMS, PMMA, COC, or any othersuitably transmissive material. The thickness of the first substratelayer 62 may range from approximately 100 μm up to approximately 1000μm. In certain preferred embodiments, the thickness of substrate layer62 may range from approximately 200 μm up to approximately 600 μm. Forexample, the thickness of substrate layer 62 may be approximately 400μm. In other preferred embodiments, the thickness of substrate layer 62may range from approximately 500 μm up to approximately 900 μm. By wayof non-limiting examples, the thickness of substrate layer 62 may beapproximately 700 μm or approximately 750 μm. In certain embodiments,the microfluidic chip 20 may be formed with only two substrate layers62, 64.

In the embodiment illustrated in FIG. 2, the microfluidic chip 20includes twenty-four micro channels 30, although, in general, any numberof micro channels 30 may be provided (e.g., as non-limiting examples, 2,4, 8, 24, 36, 72, 144, or 288 channels). According to some embodiments,when microfluidic chip 20 has twenty-four micro channels 30, themicrofluidic chip 20 may have an overall width W ranging from 70 mm to80 mm.

According to certain embodiments, each of the plurality of microchannels 30 may include a sorting or diverting mechanism 28 fordirecting particles flowing within the channels 30 into variousdownstream channels. Sorting and/or diverting may be accomplishedthrough one or more mechanisms, which may include but are not limitedto: mechanical displacement of the particle by deflecting a membranewith a piezoelectric actuator, thermal actuators, optical forcetechniques, dielectric methods (e.g., dielectrophoretic), ultrasonictransducers 27 (both bulk and/or surface), surface acoustic waveactuators, and other suitable sort mechanisms or techniques. A surfaceacoustic wave actuator may be provided as an interdigitated transducer(IDT). Exemplary ultrasonic transducers are disclosed, for example, inU.S. patent Ser. Nos. 12/631,059 and 13/818,146, the entire contents ofwhich are hereby incorporated by reference in their entirety.

The particle processing system 200 may include a receptacle or holder(not shown) for removably receiving microfluidic chip 20. Further, theparticle processing system 200 may include one or more stages forpositioning the microfluidic chip 20 relative to the optical detectionsystem 226. The stages may allow for movement (translation and/orrotation) of the microfluidic chip 20.

According to aspects of the disclosure, a microfluidic chip having amicro channel for processing a sample fluid is provided. The microchannel 30 may be in fluid communication with one or more sample inletports 410 (see FIG. 2) configured to receive a sample fluid S. Thesample inlet ports 410 may be in fluid communication with a samplereservoir, manifold, channel, well, test tube, etc. Further, the microchannel 30 may be in fluid communication with one or more focusing fluidinlet ports 450 (e.g., 450 a and 450 b) configured to receive a focusingfluid. SF. The focusing fluid inlet ports 450 may be in fluidcommunication with a sheath fluid reservoir, ma fold, channel, bag,bottle, container, etc.

According to aspects of the disclosure, the micro channel 30 may focusthe sample by using focusing fluid (e.g., sheath fluid) and a corestream forming geometry 300, for example, defined in the substrate 21 ofthe microfluidic chip 20. The core stream forming geometry 300 may beused to laminarly focus, streamline, decelerate, and/or accelerate theflow of the sample fluid S with a surrounding sheath of focusing fluidSF. In some embodiments, the core stream forming geometry 300 mayinclude a lateral fluid focusing component (see, for example, lateralfluid focusing component 432 of the embodiment of FIGS. 3A-E) and one ormore vertical fluid focusing components (see, for example, verticalfluid focusing component 434 of FIGS. 3A-E) In the context of theembodiment of FIG. 2, “lateral” may refer to a direction extendinggenerally in the plane of the substantially planar microfluidic chip 20and “vertical” may refer to a direction extending generally out of theplane of the microfluidic chip 20.

Referring now to FIGS. 3A and 3B, a portion of a micro channel 30 havinga core stream forming geometry 400 is shown. A sample fluid S flowingthrough the micro channel 30 may enter the core stream forming geometry400 along a longitudinal centerline CL (when viewed from above) of thecore stream forming geometry 400. Focusing fluid SF may enter the corestream forming geometry 400 symmetrically with respect to thelongitudinal centerline CL of the core stream forming geometry 400. Thefocusing fluid may enter the core stream forming geometry 400 at anupstream region 400 a of the core stream forming geometry and also at adownstream region of the core stream forming geometry 400 b. The samplefluid S and the focusing fluid SF may be induced to flow through themicro channel 30 via any means known in the art, including one or morepumps (e.g., peristaltic pumps), ultrasonic drivers, etc.

The core stream forming geometry 400 may include a fluid focusing region430 incorporated into a region of a flow channel 30 for generating afocused core stream flow wherein the focusing fluid SF shapes the samplestream S. The core stream forming geometry 400 is illustrated asinterior surfaces of a flow channel 30 in a microfluidic chip 20, suchas those microfluidic chips previously described. The illustrated corestream forming geometry 400 provides improved sheath flow capabilitiesand thus improved sample focusing capabilities. The core stream forminggeometry 400 may be fabricated in plastics, polycarbonate, glass,metals, or other suitable materials using microfabrication, injectionmolding, stamping, machining, 3D printing or by other suitablefabrication techniques. As such, the core stream forming geometry 400may be formed in a single substrate layer, or by a plurality of stackedlayers.

Referring to FIGS. 3A and 3B, sheath inlets ports 450 may be providedwith conical inlet shapes that are each received at a sheath aggregatingvolume 422. The sheath aggregating volumes 422 may be provided with asingle outlet or sheath fluid channel 442, or multiple outlets or sheathfluid channels to further transport focusing fluid SF to flow channel 30components. In some embodiments, there may not be any featurespecifically identifiable as a sheath aggregating volume and focusingfluid may flow directly from sheath inlet ports 450 to a focusing fluiddistribution network.

In FIGS. 3A and 3B, two sheath inlet ports 450 a, 450 b are associatedwith a single micro channel 30. Each sheath inlet port 450 may providedwith a single port outlet or sheath fluid channel 440. Sheath fluidchannel 440 a is illustrated as extending from sheath fluid inlet port450 a and sheath fluid channel 440 b is illustrated as extending fromsheath fluid inlet port 450 b. Each sheath fluid channel 440 extendsfrom an upstream region 430 a of the fluid focusing region 430 to adownstream region 430 b of the fluid focusing region 430. Each sheathfluid channel 440 is configured to transport focusing fluid SF from asheath inlet port 450 to the micro channel 20 in the downstream region430 b of the fluid focusing region 430. In the embodiment of FIGS.3A-3E, the core stream forming geometry 400 is symmetrically formedrelative to a longitudinal centerline CL of the micro channel 30 (whenviewed from above).

According to alternative embodiments, a single sheath fluid inlet port450 may be provided and a branched sheath fluid channel may beconfigured transport focusing fluid form the single sheath fluid inletport 450 to a plurality of regions of the core stream forming geometry400. Additionally, flow restrictions may be placed on one or morefluidic paths emanating from the sheath aggregating volume 422.

The fluid focusing region 430 may include a lateral fluid focusingcomponent 432 and a vertical fluid focusing component 434, both of whichmay contribute to shaping the sample stream S and increasing the axialacceleration of both the focusing or sheath fluid FS and sample Sthrough the flow channel 30. The lateral fluid focusing component mayinclude a lateral fluid focusing chamber 420. The lateral fluid focusingchamber 420 is provided with sample fluid S from a portion of the microchannel 30 in fluid communication with the sample inlet port 410.Further, the lateral fluid focusing chamber 420 is provided with sheathor focusing fluid SF from the one or more sheath fluid inlet ports 450.

According to the embodiment of FIGS. 3A-3E, the lateral fluid focusingchamber 420 is widest at its upstream end 420 a and narrowest at itsdownstream end 420 b. Between the upstream end 420 a and the downstreamend 420 b, the chamber 420 substantially linearly tapers symmetricallywith respect to the centerline CL in the lateral direction. Between theupstream end 420 a and the downstream end 420 b, the chamber 420 has asubstantially constant thickness. Further, the upstream end 420 a isprovided as a substantially flat wall having two openings, one at eachcorner, for admitting focusing fluid SF.

Thus, as illustrated, two sheath inlet ports 450 a, 450 b maysymmetrically introduce focusing fluid SF into the lateral fluidfocusing chamber 420. In FIGS. 3A-3E, a relatively short channel 442 aextends between the sheath aggregating volume 422 a and the corneropening of the lateral fluid focusing chamber 420. Similarly, arelatively short channel 442 b extends between the sheath aggregatingvolume 422 b and laterally opposed corner opening of the lateral fluidfocusing chamber 420. Thus, focusing fluid SF enters chamber 420 fromopposed lateral edges (or lateral sides) of the upstream end 420 a offocusing chamber 420.

As best shown in FIGS. 3B, 3D and 3E, at the upstream end 420 a offocusing chamber 420, a sample inlet portion 32 of the micro channel 30transporting sample fluid S extends beneath the plane of the lateralfluid focusing chamber 420. The sample inlet portion 32 of micro channel30 is centered along the longitudinal centerline CL. The sample S isinjected into the plane of the focusing chamber 42 through the openingwhere the sample inlet portion 32 of the micro channel 30 and thelateral fluid focusing chamber 420 overlap OL. As shown in FIG. 3E, thelength of the overlap OL is approximately a third of the length of thefocusing chamber 420. In other words, the sample inlet portion 32 andthe lateral fluid focusing chamber 420 share a common opening (whereotherwise they would have shared a common wall). Sample fluid S entersfocusing chamber 420 from below via a symmetrically centered openinghaving a length equal to the overlap OL region and a width equal to thewidth of micro channel 30. Thus, in this embodiment, the sample stream Sjogs from the plane of the upstream micro channel 30 upward into thefocusing fluid SF within the plane of the focusing chamber as it isintroduced into the focusing chamber.

As the sample stream and the focusing fluid progress along the lateralfluid focusing chamber 420 the lateral dimension of the chamber 420decreases. As the chamber 420 narrows or tapers in the lateral directionas the fluid travels downstream, an increasing inward force from thelateral sides of the chamber 420 acts on the fluid within the chamber,thus tending to focus (e.g., constrict) the sample S in the middle ofthe lateral fluid focusing chamber 420. The increasing inward forcefurther tends to accelerate both the sheath and the sample within thefluid focusing region 430 in the flow channel 30.

At the downstream end 420 b of the lateral fluid focusing chamber 420,the vertical fluid focusing component provides a verticalupwardly-directed focusing force. Specifically, vertical fluid focusingchannels 440 a, 440 b introduce focusing fluid FS from inlet ports 450a, 450 b into the lateral fluid focusing chamber 420 at the downstreamend 420 b. As best shown in FIGS. 3B, 3D and 3E, the vertical fluidfocusing channels 440 a, 440 b extend under channel 30. Where the topsurface of channel 440 a intersects the lower surface 30 b of channel 30an opening or aperture forms a vertical focusing flow inlet 446 so thatfocusing fluid FS from channels 440 a, 440 b may enter channel 30. Thus,the vertical fluid focusing channels 440 a, 440 b introduce focusingfluid FS into fluid focusing chamber 420 at vertical focusing flow inlet446 from below.

Referring now to FIG. 3A, 3B, 3C or 3E, the vertical fluid focusingchannels 440 a, 440 b may comprise a U-shaped or looping channel thatbranches away from the lateral fluid focusing chamber 420 and isprovided in fluid communication at aperture region 446 with the lateralfluid focusing chamber 420 further downstream. In this manner, thevertical fluid focusing channels 440 may provide a means for diverting aportion of sheath fluid that may then be reintroduced into the flowchannel 30 at a later point to focus the vertical position of the corestream of sample S.

As best shown in FIGS. 3D and 3E, the sample S enters the fluid focusingregion 430 at the upstream end 430 a in a plane P1 (see FIG. 3D) belowthe plane P2 (see FIG. 3D) in which the lateral fluid focusing chamber420 is located. The sample S is directed upward from plane P1 into theplane P2 of the lateral focusing chamber 420 in the overlapped regionOL. Then, at the downstream end 430 b of the fluid focusing region 430,the laterally focused sample within a sheath of focusing fluid (S+SF) isvertically focused upward by the introduction of focusing fluid SF atthe vertical focusing flow inlet 446 from below. The focused streamexits the fluid focusing region 430 in the P2 (see FIG. 3D) plane.

FIG. 3C is a top view of the core stream forming geometry 400, includingfluid focusing region 430 and lateral fluid focusing component 420. Asample flow S is illustrated entering the lateral focusing chamber 420from the micro channel 30. Focusing fluid flow SF is illustratedentering the lateral fluid focusing chamber 420 from each sheath inletport 450 at the upstream region 420 b of the lateral fluid focusingchamber 420. Further, the focusing fluid SF is introduced into thelateral fluid focusing chamber 420 from a lateral edge. In thisparticular embodiment, the focusing fluid SF is introduced into thelateral fluid focusing chamber 420 at a lateral, upstream corner of thefluid focusing chamber 420.

The width of the lateral fluid focusing chamber 420 decreases in adownstream direction. In this particular embodiment, the width decreaseslinearly over a majority of the fluid focusing region 430. The sheathflow SF provides an increasing shearing force on the sample S, bothaccelerating the flow of the sample S, spacing out particles in thesample, and laterally focusing the sample flow into the center of thelateral fluid focusing chamber 420.

The vertical flow of the sample S is influenced by two features of thecore stream forming geometry 400, which can be best seen in FIG. 3D.FIG. 3D represents a vertical cross-section along a longitudinal axis ofthe core stream forming geometry 400. A first downwards verticalinfluence on the sample stream is created upon entry into the lateralfluid focusing chamber 420, because the sample is introduced from underthe lateral fluid focusing region 420, so that its upward flow will beresisted by the sheath flow SF above it.

A sample flow S enters the core stream forming geometry region via microchannel 30 and via sample inlet portion 32. The sample S reaches the endof the overlapped sample inlet region OL and moves upwards against asheath flow SF in the plane of the lateral fluid focusing chamber 420.Once the core stream of sample S reaches vertical focusing flow inlet446, the vertical fluid focusing channels 440 a, 440 b introducefocusing fluid SF upward, thereby directing the sample S upwards andfocusing the sample S away from the bottom of the flow channel 30.

FIG. 3D demonstrates two notably advantageous concepts. First, therepresentative sample flow S reflects a non-perpendicular injectionpoint of the sample S, e.g., via the sample inlet portion 32. Thus, inexemplary embodiments, the sample inlet portion 32 of the micro channel30 may be configured to introduce the sample S in substantially a sameflow direction (longitudinally) as the focusing fluid SF. Second, inorder to provide enhanced core formation and centering, multiple sheathfluid inlets for introducing focusing fluid SF into fluid focusingregion 430 may be provided. For example, in a downstream focusing region420 b, vertical fluid focusing channels 440 a, 440 b may introducefocusing fluid SF at a vertical focusing flow inlet 446.

The core stream forming geometry 400 accelerates and focuses the sampleS and the sheath fluid SF around the centrally introduced sample S.Preferably, the fluid focusing region 430 focuses the sample S away fromthe sides of the micro channel. The vertically focusing component,joining the micro channel 30 downstream of the fluid focusing region430, provides additional focusing of the sample S within the focusingfluid SF. In the embodiment of FIG. 3A-3E, this secondary focusingfocuses the sample in a vertical direction from below the sample S. Thecombination of the lateral focusing and the vertical focusing providesthree-dimensional focusing of the sheath fluid around the sample.Advantageously, the resulting flow is hydrodynamically focused on allsides of the sample S away from the walls of the flow channel 30, withthe sample S being suspended as a focused core in the approximate centerof the channel 30.

After being focused in the focusing region 430, the sample may continuethrough an inspection region and a particle diverting and/or sortingregion. Further, the particles may be aligned and/or oriented accordingto specific features in the following description and a sort action maybe performed according to various mechanisms.

FIGS. 4A-4D, 5A-5D and 6A-6D introduce various embodiments which includeadditional focusing regions, e.g., tertiary focusing regions, downstreamof the secondary focusing regions.

Turning to FIGS. 4A-4D, an alternative core stream forming geometry 500is illustrated which incorporates a fluid focusing region 530. Fluidfocusing region 530 includes a vertical fluid focusing component 534configured as a double horseshoe or double loop an including first andsecond sets of vertical fluid focusing channels 540, 550. Thisembodiment relates to a core stream forming geometry 500 having a firstpair of vertical fluid focusing channel 540 a, 540 b and second pair ofvertical fluid focusing channel 550 a, 550 b configured to introduceopposing vertical fluid focusing sheath flows into lateral fluidfocusing chamber 520 for an improved core stream formation.Specifically, as best shown in FIGS. 4A and 4C, the first pair ofvertical fluid focusing channel 540 a, 540 b introduces focusing fluidSF into the downstream end 520 b of fluid focusing chamber 520 atvertical focusing flow inlet 548 (see FIG. 4C) from above. The secondpair of vertical fluid focusing channel 550 a, 550 b introduces focusingfluid SF into the downstream end 520 b of fluid focusing chamber 520 atvertical focusing flow inlet 546 (see FIG. 4C) from below. Verticalfocusing flow inlet 548 is located upstream of vertical focusing flowinlet 546. Thus, after being laterally focused, the stream is verticallyfocused downward and then vertically focused upward.

FIGS. 4A and 4C show that a sample inlet 52 (see FIG. 4A) of the microchannel 30 is positioned at the same vertical plane as the lateral fluidfocusing chamber 520. Further, the lateral fluid focusing chamber 520,the vertical fluid focusing channels 550 a, 550 b, and the sample inlet52 all lie in the same plane, plane P1 (see FIG. 4C). Additionally,vertical fluid focusing channels 540 a, 540 b lie in a plane P2 (seeFIG. 4C) above plane P1 (see FIG. 4C). After being subjected to thelaterally-directed focusing features of the lateral focusing chamber520, the vertical focusing channels 540 a, 540 b and the verticalfocusing channels 550 a, 550 b introduce opposing vertical focusingforces (via vertical focusing flow inlets 548, 546, respectively) thatact on the sample S. The focused stream exits the fluid focusing region530 in the P2 plane. Advantageously, a more focused and/or alignedsample core stream may result.

Referring to FIG. 4A, fluid focusing region 530 includes a lateral fluidfocusing component 532 which includes lateral fluid focusing chamber520. Similar to the embodiment of FIGS. 3A-3E, the lateral fluidfocusing chamber 520 is widest at its upstream end 520 a and narrowestat its downstream end 520 b. Between the upstream end 520 a and thedownstream end 520 b, the chamber 520 substantially linearly taperssymmetrically with respect to the centerline CL in the lateraldirection. Between the upstream end 520 a and the downstream end 520 b,the chamber 520 has a substantially constant thickness. Further, theupstream end 520 a is provided as a substantially flat wall having twoopenings, one at each corner, for admitting focusing fluid SF.

Thus, as illustrated, two sheath inlet ports 450 a, 450 b maysymmetrically introduce focusing fluid SF into the lateral fluidfocusing chamber 520. Referring to FIG. 4A, a relatively short channel542 a extends between the sheath aggregating volume 422 a and the corneropening of the lateral fluid focusing chamber 520. Similarly, arelatively short channel 542 b extends between the sheath aggregatingvolume 422 b and laterally opposed corner opening of the lateral fluidfocusing chamber 520. Thus, focusing fluid SF enters chamber 520 fromopposed lateral edges (or lateral sides) of the upstream end 520 a offocusing chamber 520.

In contrast to the embodiment of FIGS. 3A-3E and as best shown in FIG.4C, at the upstream end 520 a of focusing chamber 520, sample fluid Sdirectly flows into the chamber 520, in the same plane P1 in which thechamber 520 is located, via a sample inlet 52 (see FIG. 4A) of the microchannel 30.

Referring to FIG. 4B, focusing fluid SF flows into lateral fluidfocusing chamber 520 from sheath inlet ports 450. The focusing fluid SFfrom each inlet port 450 may be divided into three sheath flow portions.A first focusing fluid portion may enter the lateral fluid focusingchamber 520 at its upstream corners. In response to the narrowinglateral width of the lateral fluid focusing chamber 520, the focusingfluid SF tends to focus the sample S in the center of the lateral fluidfocusing channel 520. A second focusing fluid portion from each inletport 450 may be diverted through a vertical fluid focusing channel 550 a(or 550 b) and a third focusing fluid portion may be directed through avertical fluid focusing channel 540 a (or 540 b).

In this embodiment, the sheath aggregating volume 522 may advantageouslyprovide a greater cross sectional area than the end of the conicalsheath inlet 450, thus providing a beneficial volume for distributingfocusing fluid at relatively high sheath flow rates through each of thesheath flow portions. Further, the length the vertical focusing channels540 a, 540 b is less than the length of vertical focusing channels 550a, 550 b. The shorter length of vertical focusing channels 540 a, 540 bmeans that these channels have less resistance to flow of the focusingfluid therethrough (as compared to the vertical focusing channels 550 a,550 b). Thus, the volume of focusing fluid that may be introduced intothe fluid focusing region 530 at vertical focusing flow inlet 548 may begreater than the volume of focusing fluid that may be introduced intothe fluid focusing region 530 at vertical focusing flow inlet 546. Therelative lengths of the vertical focusing channels 540, 550 may bemodified in order control the vertical focusing of the stream. Inparticular a difference in the focusing fluid flow through the first setof vertical focusing channels 540 and the second set of verticalfocusing channels 550 may provide for an improved ability to focus thevertical position of a core stream in a flow channel 30. In general, itmay be desirable to maintain a balance between the vertical focusingforces at the vertical focusing flow inlet 548 and the vertical focusingflow inlet 546.

Turning now to FIG. 4C, a vertical cross-section along a longitudinalaxis of the core stream forming geometry 500 illustrates a core streamof sample S and a focusing fluid SF introduced into the flow channel 30at substantially the same vertical position. Focusing fluid SF from thefirst set of vertical fluid focusing channels 540 provides a downwardfocusing influence on the core stream of sample S, followed by an upwardfocusing influence from sheath fluid provided from the second set ofvertical fluid focusing channels 550. The portion of the flow channel 30following the opposing vertical sheath flows is at an elevated verticalposition relative to the lateral fluid focusing chamber 520 and thesample inlet 52. The portion of the flow channel 30 following thefocusing region may be further manipulated in a region designed toimpart orientation to particles in the core stream of sample.

FIGS. 5A-5D illustrate an alternative embodiment of the core streamforming geometry 600 having substantially the same vertical crosssection as the embodiment of FIGS. 4A-4D (compare FIG. 4C with FIG. 4C).There may be certain efficiencies gained in several stream lined aspectsrelating to the sheath fluid flow paths illustrated in FIGS. 5A-5D. Inone aspect sheath fluid passes through from each sheath aggregatingvolume 422 into a tapered focused inlet 632 which immediately puts thefocusing fluid into a trajectory for laterally focusing the core streamof sample fluid S. The tapered inlets 621 a, 621 b may eliminate anyfluid dead zone which may be caused by blunt entry geometries.

Further, the tapered inlets 621 advantageously are configured to allowthe focusing fluid SF to travel in an expanding inlet channel that sothat the focusing fluid is travelling substantially parallel (or at aslight angle) to the sample fluid S flowing in the micro channel 30immediately prior to the tapered inlets 621 merging with the channel 30.This angle may be less than 45 degrees from the longitudinal axis of themicro channel 30. In preferred embodiments, this angle may be less than30 degrees, less than 25 degrees, and even less than 20 degrees. Theinlets 621 may expand to the point of merger with the micro channel 30.The configuration of the inlets 621 provides a focusing fluid flowtrajectory that may be substantially aligned with the sample fluid flow.Notably, enabling the focusing fluid SF to expand and travelsubstantially parallel to the sample S prior to merging allows a laminarflow region to be established where all of the fluid is travelling inparallel as the fluids are merged. This streamlined merging may providea substantial reduction in fluid mixing and turbulence at the point ofmerger.

Further, the tapered inlets 621 allow the lateral fluid focusingcomponent 632 and the vertical fluid focusing component 634 to besomewhat isolated from each other. In particular, the upstream end ofthe vertical fluid focusing component is upstream of where sample Senters the fluid focusing chamber 620, thus mitigating any potential forsample S to inadvertently flow in the vertical fluid focusing component634.

In this particular embodiment, the lateral fluid focusing chamber 620has slightly convexly curved lateral edges.

Each of the first set of vertical fluid focusing channels 640 and thesecond vertical fluid focusing channels 650 are also streamlined with acommon inlet 655. However, in contrast to the embodiment of FIGS. 4A-4D,in this embodiment, the cross-sectional areas of the vertical focusingchannels 650, 640 need not be constant along their length, but may varyfrom one portion to another. Further, the cross-sectional area ofvertical fluid focusing channels 640 may be larger than the flattercross-section area of vertical fluid focusing channels 650. This largercross-sectional area of vertical fluid focusing channels 640 relative tothe flatter cross section of vertical fluid focusing channels 650 mayallow a greater flow of vertical focusing fluid to enter chamber 620 atvertical focusing flow inlet 648 (see FIG. 5C) than at vertical focusingflow inlet 646 (see FIG. 5C).

The greater cross-sectional area and the shorter length of verticalfocusing channels 640 a, 640 b mean that these channels have lessresistance to flow of the focusing fluid therethrough (as compared tothe vertical focusing channels 650 a, 650 b). Thus, the volume offocusing fluid that may be introduced into the fluid focusing region 630at vertical focusing flow inlet 648 may be greater than the volume offocusing fluid that may be introduced into the fluid focusing region 630at vertical focusing flow inlet 646. The relative cross-sectional areasand/or the relative lengths of the vertical focusing channels 640, 650may be modified in order control the vertical focusing of the stream. Insome aspects, it may be desirable to maintain a balance between thevertical focusing forces at the vertical focusing flow inlet 548 and thevertical focusing flow inlet 546. Thus, providing varying lengths,cross-sectional areas and/or non-constant cross-sectional areas for thedifferent vertical fluid focusing channels may allow the verticalfocusing forces to be balanced.

Thus, advantageously, aspects disclosed herein allow the designer totailor the focusing flows acting on the stream so as to optimize theposition and/or shape of the focused stream within the channel.

FIGS. 6A-6D illustrate another embodiment of the core stream forminggeometry 700. Similar to the embodiment of FIGS. 5A-5D and as best shownin FIG. 6A, this embodiment also has streamlined fluid focusing flowcomponents, such as a dedicated tapered inlet 721 extending into thelateral fluid focusing chamber 720 from the inlet port 450 and a commonfocusing fluid channel 755 connected directly to the sheath aggregatingvolume 422 of each sheath inlet 450 and supplying focusing fluid SF tothe first and second sets of vertical fluid focusing channels 740, 750.Additionally, FIGS. 6A-6D illustrate an alternative vertical placementof some portions of each of the first vertical fluid focusing channel740 and the second vertical fluid focusing channel 750.

Further, compared to the embodiment of FIGS. 5A-5D, the embodiment ofFIGS. 6A-6D are provided with a relatively large cross sectional areasof both the first set of vertical fluid focusing channels 740 and thesecond set of vertical fluid focusing channels 750. This greatercross-sectional area provides less resistance to the focusing fluidentering the vertical fluid focusing component 734 relative to thefocusing fluid enter the lateral fluid focusing component 732. Thus,another way to balance and/or control the focusing forces acting on thesample S is provided by controlling the relative fluidic resistances ofthe focusing fluid flow into the vertical fluid focusing component 734and into the lateral fluid focusing component 732.

Even further, the embodiment of FIGS. 6A-6D are provided with anenhanced sheath aggregating volume 422 in other to accommodate therelatively large cross sectional areas of both the first set of verticalfluid focusing channels 740 and the second set of vertical fluidfocusing channels 750. Also of interest is that the vertical fluidfocusing channels 740, 750 are configured with a reduced downstreamcross-sectional area (as compared to the greater cross-sectional areaprovided in the upstream portion of the channels).

Referring back to FIG. 2 and also to FIGS. 3D, 4C, 5C and 6C, accordingto certain embodiments, substrate 21 may be formed by bonding orotherwise attaching an upper substrate layer 62 to a lower substratelayer 64. Referring now to FIG. 7A, a top view of the substrate 21 isshown with the core stream forming geometry 300 visible through the toplayer of the substrate. FIG. 7B, a lower surface 62 b of the uppersubstrate 62 of the substrate of FIG. 7A is shown. Portions of the fluidfocusing components 300′ of the core stream forming geometry 300 areshown provided in the lower surface 62 b. In FIG. 7B, a lower surface 62b of the upper substrate 62 of the substrate of FIG. 7A is shown.Complementary portions of the fluid focusing components 300″ core streamforming geometry 300 are shown provided in the lower surface 62 b. InFIG. 7C, an upper surface 64 a of the lower substrate 64 of thesubstrate of FIG. 7A is shown. Portions of the fluid focusing componentsare shown provided in the upper surface 64 a. The portions of the fluidfocusing components 300′, 300″ provided in these substrate layersurfaces may be provided (via additive or subtractive manufacturing).When the upper surface 64 a and the lower surface 62 b are assembledtogether with the complementary portions of the fluid focusingcomponents aligned with each other, the complete core stream forminggeometry is formed. Thus, a complicated core stream forming geometry 300such as the exemplary core stream forming geometries described herein,may be simply and efficiently provided with just two substrate layers.While the core stream forming geometry 300 depicted in the embodiment inFIGS. 7A-C is illustrated as the exemplary core stream forming geometry600 of the embodiment of FIGS. 5A-D, it is appreciated that an uppersubstrate layer 62 and a lower substrate layer 64 may similarly be usedto define any number of different stream forming geometries including,for example, any of the exemplary stream forming geometries 400, 500,600 and 700 described herein, for example, with respect to theembodiments of FIGS. 3A-E, 4A-D, 5A-D and 6A-D.

As can be understood from the foregoing, features described for focusinga core stream may be combined with various features for monitoring,detecting, analyzing, and/or sorting particles of interest. See, e.g.,U.S. Pat. Nos. 6,877,528, 6,808,075, and 7,298,478, which are herebyincorporated by reference in their entireties.

A system and method for producing a focused sample in a flow channel,such as a micro channel, has been described herein. As can be easilyunderstood from the foregoing, the basic concepts of the presentdisclosure may be embodied in a variety of ways. As such, the particularembodiments or elements disclosed by the description or shown in thefigures accompanying this application are not intended to be limiting,but rather illustrative of the numerous and varied embodimentsgenerically encompassed by the present disclosure or equivalentsencompassed with respect to any particular element thereof. In addition,the specific description of a single embodiment or element may notexplicitly describe all embodiments or elements possible; manyalternatives are implicitly disclosed by the description and figures.

Moreover, for the purposes of the present disclosure, the term “a” or“an” entity refers to one or more of that entity. As such, the terms “a”or “an”, “one or more” and “at least one” can be used interchangeablyherein.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. It will beunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

We claim:
 1. A microfluidic assembly for use with a particle processinginstrument, the microfluidic assembly comprising: a substrate; and aflow channel formed in the substrate, the flow channel having: an inletconfigured to receive a sample stream; a fluid focusing regionconfigured to focus the sample stream, the fluid focusing region havinga lateral fluid focusing feature to focus the sample stream laterallyand first and second vertical fluid focusing features to focus thesample stream vertically after the sample stream is focused laterally,wherein a bottom surface of the flow channel lies in a first planeupstream of the first and second vertical fluid focusing features andthe bottom surface of the flow channel shifts vertically upward to liein a second plane downstream of the first and second vertical fluidfocusing features, the sample stream and a focusing fluid entering thefluid focusing region in a same plane; and an inspection region at leastpartially downstream of the fluid focusing region.
 2. The microfluidicassembly of claim 1, wherein the lateral, the first vertical, and thesecond vertical fluid focusing features are provided at differentlongitudinal locations along the flow channel.
 3. The microfluidicassembly of claim 2, wherein each of the fluid focusing featuresintroduces a focusing fluid into the flow channel at the differentlongitudinal locations along the flow channel.
 4. The microfluidicassembly of claim 1, wherein the lateral fluid focusing feature isconfigured to introduce focusing fluid into the flow channelsymmetrically with respect to a centerline of the sample stream.
 5. Themicrofluidic assembly of claim 4, wherein the first and second verticalfluid focusing features are located downstream of the lateral fluidfocusing feature; wherein the first vertical fluid focusing featureincludes a first vertical fluid focusing aperture configured tointroduce focusing fluid into the flow channel from above the samplestream; and wherein the second vertical fluid focusing feature includesa second vertical fluid focusing aperture configured to introducefocusing fluid into the flow channel from below the sample stream. 6.The microfluidic assembly of claim 5, wherein the first vertical fluidfocusing aperture is in fluid communication with a first vertical fluidfocusing channel and the second vertical fluid focusing aperture is influid communication with a second vertical fluid focusing channel. 7.The microfluidic assembly of claim 5, wherein the first vertical fluidfocusing aperture is in fluid communication with a first pair of fluidfocusing channels; wherein the second vertical fluid focusing apertureis in fluid communication with a second pair of fluid focusing channels;and wherein each of the first pair and the second pair of fluid focusingchannels are symmetrically arranged with respect to a centerline of theflow channel.
 8. The microfluidic assembly of claim 1, wherein thelateral fluid focusing feature comprises an acoustic wave transducer forproducing pressure waves in the focusing region of the flow channel. 9.The microfluidic assembly of claim 1, further comprising a divertingmechanism in communication with the flow channel, wherein the divertingmechanism includes a bubble valve, a piezoelectric actuator, a thermalactuator, or an acoustic wave actuator.
 10. The microfluidic assembly ofclaim 1, wherein the fluid focusing region has a varying width upstreamof the first and second vertical focusing fluid features; and whereinthe flow channel has a constant width between the first and secondvertical focusing fluid features and the inspection region.
 11. Themicrofluidic assembly of claim 1, wherein within the fluid focusingregion the flow channel transitions from a first cross section shape toa second cross section shape different from the first cross sectionshape.
 12. The microfluidic assembly of claim 1, wherein each of thefluid focusing features is in fluid communication with a first focusingfluid inlet port provided on a top surface of the substrate.
 13. Themicrofluidic assembly of claim 1, wherein each of the fluid focusingfeatures is in fluid communication with a pair of focusing fluid inletports provided on a top surface of the substrate.
 14. A microfluidicassembly for use with a particle processing instrument, the microfluidicassembly comprising: a substrate; and a flow channel formed in thesubstrate, the flow channel having: an inlet configured to receive asample stream; and a fluid focusing region configured to focus thesample stream, the fluid focusing region having a lateral fluid focusingfeature, a first vertical fluid focusing feature adjacent to the lateralfluid focusing feature, and a second vertical fluid focusing featuredisposed downstream of the first vertical fluid focusing feature,wherein a top surface of the flow channel lies in a first plane upstreamof the first and second vertical fluid focusing features and the topsurface of the flow channel shifts vertically upward to lie in a secondplane downstream of the first and second vertical fluid focusingfeatures.
 15. The microfluidic assembly of claim 14, wherein thelateral, the first vertical, and the second vertical fluid focusingfeatures are provided at different longitudinal locations along the flowchannel.
 16. The microfluidic assembly of claim 14, wherein the lateralfluid focusing feature is configured to introduce focusing fluid intothe flow channel symmetrically with respect to a centerline of thesample stream.
 17. A microfluidic assembly for use with a particleprocessing instrument, the microfluidic assembly comprising: asubstrate; and a flow channel formed in the substrate, the flow channelhaving: an inlet configured to receive a sample stream; a fluid focusingregion configured to focus the sample stream, the fluid focusing regionhaving a lateral fluid focusing feature, a first vertical fluid focusingfeature adjacent to the lateral fluid focusing feature, and a secondvertical fluid focusing feature downstream of the first vertical fluidfocusing feature, wherein a bottom surface of the flow channel lies in afirst plane upstream of the first and second vertical fluid focusingfeatures and the bottom surface of the flow channel shifts verticallyupward to lie in a second plane downstream of the first and secondvertical fluid focusing features, the sample stream and a focusing fluidentering the fluid focusing region in a same plane; and an inspectionregion at least partially downstream of the fluid focusing region.
 18. Amicrofluidic assembly for use with a particle processing instrument, themicrofluidic assembly comprising: a substrate; and a flow channel formedin the substrate, the flow channel having: an inlet configured toreceive a sample stream; a fluid focusing region configured to focus thesample stream, the fluid focusing region having a lateral fluid focusingfeature, a first vertical fluid focusing feature disposed downstream ofthe lateral focusing feature, and a second vertical fluid focusingfeature disposed downstream of the first vertical fluid focusingfeature, wherein a bottom surface of the flow channel lies in a firstplane upstream of the first and second vertical fluid focusing featuresand the bottom surface of the flow channel shifts vertically upward tolie in a second plane downstream of the first and second vertical fluidfocusing features, the sample stream and a focusing fluid entering thefluid focusing region in a same plane; and an inspection region at leastpartially downstream of the fluid focusing region.